Therapeutic targeting of CNBP phase separation inhibits ribosome biogenesis and neuroblastoma progression via modulating SWI/SNF complex activity

Abstract Background Neuroblastoma (NB) is the most common extracranial malignancy in childhood; however, the mechanisms underlying its aggressive characteristics still remain elusive. Methods Integrative data analysis was performed to reveal tumour‐driving transcriptional regulators. Co‐immunoprecipitation and mass spectrometry assays were applied for protein interaction studies. Real‐time reverse transcription‐polymerase chain reaction, western blotting, sequential chromatin immunoprecipitation and dual‐luciferase reporter assays were carried out to explore gene expression regulation. The biological characteristics of NB cell lines were examined via gain‐ and loss‐of‐function assays. For survival analysis, the Cox regression model and log‐rank tests were used. Results Cellular nucleic acid‐binding protein (CNBP) was found to be an independent factor affecting NB outcome, which exerted oncogenic roles in ribosome biogenesis, tumourigenesis and aggressiveness. Mechanistically, karyopherin subunit beta 1 (KPNB1) was responsible for nuclear transport of CNBP, whereas liquid condensates of CNBP repressed the activity of switch/sucrose‐nonfermentable (SWI/SNF) core subunits (SMARCC2/SMARCC1/SMARCA4) via interaction with SMARCC2, leading to alternatively increased activity of SMARCC1/SMARCA4 binary complex in facilitating gene expression essential for 18S ribosomal RNA (rRNA) processing in tumour cells, extracellular vesicle‐mediated delivery of 18S rRNA and subsequent M2 macrophage polarisation. A cell‐penetrating peptide blocking phase separation and interaction of CNBP with SMARCC2 inhibited ribosome biogenesis and NB progression. High KPNB1, CNBP, SMARCC1 or SMARCA4 expression or low SMARCC2 levels were associated with poor survival of NB patients. Conclusions These findings suggest that CNBP phase separation is a target for inhibiting ribosome biogenesis and tumour progression in NB via modulating SWI/SNF complex activity.

whereas liquid condensates of CNBP repressed the activity of switch/sucrosenonfermentable (SWI/SNF) core subunits (SMARCC2/SMARCC1/SMARCA4) via interaction with SMARCC2, leading to alternatively increased activity of SMARCC1/SMARCA4 binary complex in facilitating gene expression essential for 18S ribosomal RNA (rRNA) processing in tumour cells, extracellular

BACKGROUND
As the most common extracranial malignancy arising from neural crest, neuroblastoma (NB) occurs mainly in adrenal medulla or sympathetic ganglia during childhood 1 and is characterised by striking heterogeneity of clinical and biological features. 1 Currently, NB cases are stratified according to age at diagnosis, histopathological presentation, MYCN amplification, disease stage, DNA ploidy or chromosomal aberrations. 1,2 Despite rigorous chemotherapy, radiotherapy or surgical treatment, the prognosis of NB patients in high-risk group is still dismal, with longterm survival rates fewer than 40%. 1 Thus, it is urgent to identify targets driving tumourigenesis and aggressiveness of NB, which is important for risk stratification and accurate therapeutics of patients. Cellular nucleic acid-binding protein (CNBP) is a conserved protein of 19 kDa, 3 while amplification of its CCTG repeat within intron 1 leads to myotonic dystrophy type 2, an autosomal dominant multisystem disease. 4 As a nucleic acid chaperone, CNBP participates in negative or positive regulation of gene transcription, such as Wnt 5 5 and interleukin 6. 6 In gastric cancer, CNBP facilitates the growth and aggressive behaviours of cancer cells via up-regulating human antigen R at the transcriptional levels. 7 Additionally, CNBP facilitates the progression of melanoma and lung cancer by directly activating promoters of matrix metallopeptidase 2, matrix metallopeptidase 14 or E2F transcription factor 2. 8 Meanwhile, through translational up-regulation of ornithine decarboxylase, a regulator of polyamine synthesis, CNBP promotes the growth of medulloblastoma. 9 However, the action modes and regulatory mechanisms of CNBP during NB progression are yet unknown.
In the current study, CNBP is discovered as an independent factor affecting NB outcome. CNBP exerts oncogenic roles in ribosome biogenesis, tumourigenesis and aggressiveness. Karyopherin subunit beta 1 (KPNB1) is responsible for nuclear transport of CNBP. By forming liquid condensates together with switch/sucrosenonfermentable (SWI/SNF)-related matrix-associated actin-dependent regulator of chromatin (SMARC) subfamily c member 2 (SMARCC2), CNBP represses the activity of SMARCC2/SMARC subfamily c member 1 (SMARCC1)/SMARC subfamily a member 4 (SMARCA4), core subunits of mammalian SWI/SNF chromatinremodelling complex, leading to alternatively increased activity of SMARCC1/SMARCA4 binary complex in facilitating gene expression essential for 18S ribosomal RNA (rRNA) processing in tumour cells, which further activates M2 macrophage polarization via extracellular vesicle (EV)mediated 18S rRNA delivery. Pharmacological or genetic inhibition of KPNB1/CNBP and cell-penetrating peptide blocking phase separation and interaction of CNBP with SMARCC2 are efficient in reducing tumourigenesis and aggressiveness. Clinical NB cases with up-regulation of KPNB1, CNBP, SMARCC1, SMARCA4 or down-regulation of SMARCC2 have poor survival and prognosis, indicating KPNB1/CNBP/SMARCC2 axis as a valuable target for therapeutics of tumours.

CNBP is linked to poor prognosis and clinical progression of NB
For discovering important transcriptional regulators of NB, we first analysed the differentially expressed transcriptional regulators in 249 (TARGET) NB cases with varied status of age (<18 months vs. >18 months), MYCN amplification (with vs. without), clinical stages of International Neuroblastoma Staging System (INSS, 1+2+4S vs. 3+4) or risk classification (low vs. high). As shown in Figure 1A, 18 transcriptional regulators were noted to be consistently associated with older age at diagnosis (>18 months), MYCN amplification, advanced INSS stage and high risk in 249 NB cases (Table S1). Similarly, we found 229 transcriptional regulators associated with these features in another independent cohort of 498 (GSE62564) NB cases ( Figure 1A and Table S1). By overlapping analysis of results derived from these two independent cohorts, 18 transcriptional regulators were identified as candidates essential for aggressiveness of NB ( Figure 1A). By analysing their survival significance, only CNBP was found to be associated with poor survival in 249 (TARGET), 498 (GSE62564), 144 (gencode19), 102 (GSE3446) and 88 (GSE16476) NB patients ( Figure 1A and Table S1). In 113 NB cases derived from the Oncogenomics database (Table S2), high copy number of CNBP gene was substantially related to low event-free survival probability (P = 1.4 × 10 −2 , Figure 1B). The CNBP levels were linked to worse alive status in 249 (TARGET, P = 3.1 × 10 −2 ), 498 (GSE62564, P = 1.5 × 10 −17 ), 144 (gen-code19, P = 1.7 × 10 −4 ), 102 (GSE3446, P = 1.5 × 10 −2 ) and 88 (GSE16476, P = 2.4 × 10 −2 ) patients suffering from NB (Figures 1C and S1A), while worse survival was observed in patients with up-regulation of both MYCN and CNBP ( Figure 1D). Elevated CNBP expression was detected in NB specimens or cultured cell lines ( Figure 1E,F). There was up-regulation of Cnbp in hyperplastic sympathetic ganglia of TH-MYCN transgenic mice ( Figure S1B). Notably, ectopic expression of MYCN led to up-regulation of CNBP in both MYCN-amplified and non-amplified cells (IMR-32 and SH-SY5Y, Figure S1C). Although knockdown of MYCN decreased the levels of CNBP in MYCN-amplified IMR-32 cells ( Figure S1C), CNBP expression was not associated with that of MYCN in NB cell lines without MYCN amplification ( Figure 1F). Moreover, MYCN levels were unaffected by CNBP within these NB cells ( Figure  S1C). In multivariate analysis, CNBP expression served as an independent prognostic indicator in 498 (GSE62564) NB cases (Table S3). Immunohistochemical staining indicated higher nuclear CNBP expression in NB specimens with poor differentiation, accompanied by more nucleolar organiser region (NOR) dots, when compared to those with good differentiation (Figures S1D and 1G). These results suggested that CNBP was related to poor prognosis and clinical progression of NB.

CNBP facilitates aggressive behaviours of NB cells
Based on above CNBP expression profiling, SH-SY5Y, IMR-32 (with middle CNBP levels) and BE(2)-C (with high CNBP levels) cells were selected as models for further biological studies. Within these NB cells, the expression of CNBP was up-or down-regulated by steady transfection of CNBP construct, distinct short hairpin RNAs (shRNAs) against CNBP (sh-CNBP), or nuclease-dead clustered regularly interspaced short palindromic repeatassociated protein 9 (dCas9) vectors 10 (Figures 2A and  S2A,B). The viability of NB cells was enhanced or diminished by over-expression or silencing of CNBP ( Figure 2B), accompanied by increase or decrease in 40S ribosomal subunit assembly, respectively ( Figure S2C). In SH-SY5Y cells persistently over-expressing CNBP, NOR dots and protein synthesis were increased ( Figure S2D,E). Notably, silencing of CNBP-induced apoptosis of SH-SY5Y (wildtype p53) cells, without significant changes in cell cycle phases ( Figure S2F,G). Meanwhile, there was significant G 2 /M cell cycle arrest, but no changes of apoptosis, in BE(2)-C (mutant p53) cells with steady transfection of sh-CNBP ( Figure S2F,G), which was consistent with previous findings that inhibition of ribosome biogenesis induces apoptosis or G 2 /M arrest in wild-type or mutant p53 NB cells, respectively. 11 In addition, in vitro proliferative and invasive behaviours of SH-SY5Y and IMR-32 cells were also increased or repressed upon steady transfection of CNBP or sh-CNBP, respectively ( Figure 2C). Consistently, steady upor down-regulation of CNBP caused a significant increase or decrease in volume, weight, proliferative index and microvessel density of xenograft tumours generated by subcutaneous injection of SH-SY5Y cells into nude mice ( Figures 2D,E and S2H). Meanwhile, intravenous administration of NB cells with steady CNBP over-expression resulted in more lung metastases and worse survival of nude mice ( Figure 2F). Conversely, nude mice treated with NB cells stably knocking down CNBP presented fewer lung metastatic colonies and higher survival possibility ( Figure 2F). These findings revealed that CNBP facilitated aggressive behaviours of NB cells.

KPNB1 facilitates nuclear translocation of CNBP to interact with SMARCC2 in liquid condensates
To determine CNBP-binding partners, CNBP antibody pulled down proteins from cellular lysates were subjected  (Figures 3A and  S3A and Table S4). There were 88 proteins binding to endogenous CNBP in IMR-32 cells, 82 of which were consistently detected following stable transfection of CNBP ( Figure 3A). Among them, KPNB1 was essential for nucleocytoplasmic transport ( Figure 3A), while SMARCC2 was one subunit of SWI/SNF complex ( Figure 3B). Notably, high KPNB1 expression (P = 2.1 × 10 −2 and 1.0 × 10 −14 ) or low SMARCC2 levels (P = 2.4 × 10 −2 and 6.9 × 10 −11 ) were linked to poor outcome of NB patients in public datasets (TARGET and GSE62564, Figure 3A,B). Endogenous physical interaction of CNBP with KPNB1 or SMARCC2 protein was validated in NB cells ( Figure S3B). Additionally, bimolecular fluorescence complementation (BiFC) experiment revealed that in IMR-32 cells, CNBP interacted with either KPNB1 or SMARCC2, while no interaction was observed between KPNB1 and SMARCC2 ( Figure 3C). Without affecting the expression of CNBP or SMARCC2, ectopic expression of KPNB1 boosted nuclear translocation of CNBP in NB cells ( Figures 3D and S3C). Meanwhile, genetic knockdown or pharmacological inhibition of KPNB1 using importazole (IPZ) 12 led to cytoplasmic accumulation of CNBP in IMR-32 cells (Figures 3D-F and S3D). In particular, IPZ treatment reduced the increase in proliferation and invasion of NB cells induced by CNBP activation (Figures 3G and S3E).
Since there were also SMARCA4 and SMARCC1, established SWI/SNF core subunits ( Figure S4A), in proteins pulled down by CNBP antibody from IMR-32 cells stably over-expressing CNBP ( Figure 3B), we further investigated the interaction of CNBP with these SWI/SNF subunits. Steady transfection or knockdown of CNBP promoted or inhibited the interaction of CNBP with SMARCC2, SMARCC1 or SMARCA4 in SH-SY5Y and IMR-32 cells ( Figures 4A and S4B). When SMARCC2 was over-expressed or knocked down, the interaction of CNBP with SMARCC1 or SMARCA4 in NB cells was increased or attenuated (Figures 4B,C and S4C,D). The physical interaction between CNBP and SMARCC2 was unaffected by silencing of SMARCC1 or SMARCA4 ( Figures 4C and S4E). Alternatively, SMARCC2 silencing promoted the interaction of SMARCA4 with SMARCC1 in NB cells ( Figure 4C). Notably, arginine-glycine-glycine (RGG) domain (22-42 amino acids [aa]), but not zinc finger domains, of CNBP protein mediated its binding to SMARCC2 ( Figure S4F,G). Meanwhile, SANT domain (596-647 aa), rather than Swirm domain (424-521 aa) or C170 domain (647-1215 aa), within SMARCC2 protein was required for its interaction with CNBP, which was also demonstrated by in vitro binding test using glutathione S-transferase (GST)-tagged CNBP or His-tagged SMARCC2 proteins ( Figure S4F,G). Since RGG domain of CNBP protein was discovered to locate within intrinsically disordered regions (IDRs) via Preditor of Natural Disordered Regions program 13 ( Figure 4D), the potential liquid-liquid phase separation (LLPS) of CNBP and SMARCC2 protein was further investigated. Fluorescence imaging assay indicated that recombinant CNBP-mCherry and SMARCC2-EGFP proteins (purity > 90%) assembled into condensates in vitro, with similar compartmentation in SH-SY5Y cells, while deletion of IDR abolished the LLPS of CNBP in vitro and in vivo ( Figure 4E). To explore their liquid-like features, fluorescence recovery after photobleaching (FRAP) assay was performed, which indicated rapid exchange kinetics of CNBP and SMARCC2 within condensates ( Figure 4F,G). These data indicated that KPNB1 facilitated nuclear translocation of CNBP to interact with SMARCC2 in liquid condensates.

2.4
Phase separation of CNBP modulates SWI/SNF activity to facilitate gene expression essential for 40S ribosomal subunit assembly To determine target genes of CNBP or SMARCC2, RNA sequencing revealed that ectopic expression of CNBP led to significant expression alteration of 3524 genes in IMR-32 cells ( Figure 5A and Table S5). In chromatin immunoprecipitation sequencing (ChIP-seq) assay, there  were 6389 and 295 genes with endogenous SMARCC2 or CNBP-binding peaks, respectively ( Figure 5B). Based on comprehensive analysis of possible action modes, 954 genes were identified as top ranking targets uniquely recognised by SMARCC2, with CNBP acting as a cofactor ( Figures 5C and S5A). In three separate datasets (GSE62564, GSE45547, GSE16476), 124 of them had consistent relationship with NB patients survival ( Figures 5C and  S5B), while DAVID analysis (https://david-d.ncifcrf.gov) revealed ribosome biogenesis as an important involved pathway ( Figure 5D and Table S6), mainly participated by bystin like (BYSL), NOP58 ribonucleoprotein (NOP58) and ribosomal RNA processing 9 (RRP9, Figure 5E). Notably, ectopic expression of CNBP or its IDR-deficient form abolished or facilitated the enrichment of SMARCC2 on these target genes ( Figure S5C), resulting in elevated or decreased promoter activity and transcript levels of BYSL, NOP58 and RRP9, respectively ( Figure S5D,E). To further reveal the effects of CNBP and SWI/SNF subunits on target gene expression, sequential ChIP and quantitative PCR (qPCR) assays were performed using first antibody specific for SMARCC2, SMARCC1 or SMARCA4, respectively. Ectopic expression of CNBP reduced the enrichment of SMARCC2/SMARCC1/SMARCA4 subunits, but increased the alternative binding of SMARCC1/SMARCA4 binary complex, on promoter regions of BYSL, NOP58 and RRP9, which was abolished by transfection of SMARCC2 ( Figures 5F and S5F). Notably, knockdown of SMARCC1 or SMARCA4 attenuated the up-regulation of target genes induced by ectopic CNBP expression ( Figure S5G,H). In public datasets of 249 (TARGET) and 498 (GSE62564) NB patients, elevated levels of SMARCC1 or SMARCA4 were linked to worse survival ( Figure S5I). Accordingly, upregulation of SMARCC2 caused a decrease in promoter activation, transcript levels, as well as protein expression of BYSL, NOP58 or RRP9 in IMR-32 cells, which was reversed by over-expression of CNBP ( Figure 5G-I). Rather, the levels of previously recognised SWI/SNF target genes c-Myc or cyclin D1 [14][15][16] were not affected by CNBP, SMARCC2, SMARCC1 or SMARCA4 in SH-SY5Y cells (Figures S5J,K and S12). In addition, knockdown of BYSL, NOP58 or RRP9 restored the alteration in their expression and aggressive features of NB cells following CNBP activation ( Figure  S6A-E).
Consistently, persistent over-expression of CNBP facilitated the 18S rRNA processing from 18S-E pre-rRNA in IMR-32 cells ( Figure 6A), accompanied by increased abundance of 40S ribosomal subunit, 80S ribosomal subunit and polysomes ( Figure 6B). Meanwhile, ectopic expression of SMARCC2 restored the changes in 18S rRNA levels, ribosome biogenesis, NORs dots and protein synthesis induced by CNBP ( Figures 6A-E and S7A). Treatment with CX-5461, an established inhibitor of ribosome biogenesis, 17 repressed the ribosomal subunit assembly ( Figure 6F), and rescued the increase in NORs dots, nascent protein production and protein synthesis of NB cells induced by CNBP ( Figures 6G-I and S7A). Stable over-expression of CNBP caused an increase in proliferative or invasive features of NB cells, which was prevented upon SMARCC2 transfection or CX-5461 treatment (Figures 6J,K and S7B,C). These results suggested that phase separation of CNBP modulated SWI/SNF activity to facilitate gene expression essential for 40S ribosomal subunit assembly.

CNBP exerts oncogenic functions via repressing SMARCC2 activity
Since immunohistochemical staining revealed the enrichment of F4/80 + M2 macrophages in subcutaneous xenograft tumours formed by CNBP over-expressing NB cells ( Figure S7D), the impacts of CNBP or SMARCC2 on interplay between NB and macrophages were further explored. During co-culture studies, NB cells stably overexpressing SMARCC2 inhibited the polarization of M2 macrophages from monocyte cell line Tohoku Hospital Pediatrics-1 (THP-1), while ectopic expression of CNBP rescued this alteration ( Figure 7A). We further extracted EVs from culture medium of NB cells, and validated them via electron microscopic analysis ( Figure 7B), particle size assay ( Figure S7E) and surface marker (CD9 and CD63) detection ( Figure 7C). The 18S rRNA levels were reduced in EVs isolated from NB cells stably over-expressing SMARCC2, however, ectopic expression  of CNBP restored this change ( Figure 7C). The NB cells' ability to transmit Dil-labelled EVs to macrophages was observed via immunofluorescence assay ( Figure 7D). In M2 macrophage co-cultured with IMR-32 cells stably overexpressing SMARCC2, there was a substantial reduction in levels of transforming growth factor beta-1 (TGFB1) and interleukin-10 (IL-10), which was reversed by transfection of CNBP ( Figure 7E). As a return, M2 macrophage culture medium significantly stimulated the viability, proliferation and invasive behaviours of NB cells ( Figure S7F-H). Steady transfection of SMARCC2 led to decrease in volume, weight, proliferative index, microvessel density and M2 macrophage accumulation of subcutaneous tumours produced by IMR-32 cells in immune-deficient mice, and these effects were eliminated by transfection of CNBP ( Figures 7F and S8A,B). Meanwhile, there were fewer lung metastatic counts and higher survival possibility of nude mice after intravenous injection of IMR-32 cells with steady SMARCC2 over-expression, which were reversed by CNBP transfection (Figures 7G and S8C). These findings showed that CNBP suppressed SMARCC2 activity to exert its oncogenic effects.

Targeting phase separation and interaction of CNBP with SMARCC2 inhibits NB progression
Considering the significance of RGG region for interaction of CNBP with SMARCC2, we prepared cell-penetrating peptides by using the Peptiderive website, 18 which were designated as CNBP inhibitory peptides with a length of 12 aa (CIP-12, Figure 8A). CIP-12, but not the mutant control peptide (CIP-12 Mut), pulled down endogenous CNBP from BE(2)-C cell lysates ( Figure 8B). There was accumulation of peptides within the nucleus of NB cells treated with CIP-12, but not with CIP-12 Mut ( Figure 8C). The phase separation of CNBP, but not of SMARCC2, was abolished by CIP-12 treatment ( Figure 8D). In BE(2)-C cells, CIP-12 treatment abolished endogenous interaction of CNBP with SMARCC2 ( Figure 8E), resulting in alteration of downstream genes ( Figure 8F). CIP-12 administration reduced the viability of BE(2)-C cells, rather than affecting that of non-transformed cells ( Figure  S9A). Additionally, CIP-12 reduced the ribosomal subunit assembly, NOR dots, protein synthesis, proliferation and invasiveness of NB cells ( Figures S9B-F and 8G). Intravenous administration of CIP-12, but not CIP-12 Mut, reduced the volume, weight, downstream gene expression, Ki-67 levels, CD31-positive microvessels, NOR dot number and M2 macrophage accumulation of BE(2)-C-formed subcutaneous tumours ( Figures 8H,I and S10A). Intravenous treatment of CIP-12 into nude mice also decreased the lung metastasis generated by BE(2)-C cells, and also prolonged mice survival time ( Figures 8H,I and S10B). In two public NB datasets (GSE62564 and GSE45547), BYSL (P = 1.0 × 10 −17 and 1.8 × 10 −9 ), NOP58 (P = 3.4 × 10 −13 and 2.9 × 10 −8 ) or RRP9 (P = 4.6 × 10 −14 and 2.0 × 10 −13 ) expression was substantially linked to worse survival of patients ( Figure S11). Above all, these findings showed that blocking phase separation and interaction of CNBP with SMARCC2 inhibited NB progression.

DISCUSSION
Ribosome biogenesis is a biological process for translating mRNA into protein. 19 Cells with vigorous growth and metabolism usually have large nucleoli and active rRNA synthesis, 19 while hyper-activation of ribosome biogenesis is crucial for the development and spread of tumours. 20 Meanwhile, tumour suppressor p53 is activated upon inhibition of ribosome biogenesis. 11,21 Herein, we discovered CNBP as an independent factor affecting NB outcome by driving ribosome biogenesis. Although MYCN facilitated the levels of CNBP in MYCN-amplified NB cells, the mechanisms regulating high CNBP expression in MYCNnon-amplified NB warrant further investigation. We found that knockdown of CNBP inhibited ribosome biogenesis, leading to apoptosis or cell cycle G 2 /M arrest in  wild-type or mutant p53 NB cells. Since lung metastasis is linked to a poor prognosis of NB patients, 22 the effects of CNBP on aggressiveness of NB were investigated in a mouse lung metastasis model. During the study, we observed no obvious metastasis in the liver, kidney or bone marrow. However, in severe combined immunodeficiency (SCID)-Beige mice, vein tail injection of NB cells results in metastasis in kidney, lung and liver. 23 We believe that this difference may be due to varied immune microenvironment in host that determines the survival of tumour cells in distant organs.
Previous studies have shown that CNBP promotes structure rearrangement of nucleic acids in an adenosine triphosphate (ATP)-independent way, and regulates gene expression at the transcriptional or posttranscriptional levels. 3 Unexpected, our results indicate that CNBP serves as a modulator of SWI/SNF subunit activity via interacting with SMARCC2. We demonstrate that nuclear transport of CNBP is mediated by KPNB1, whereas phase separation of CNBP inhibits the activity of SMARCC2/SMARCC1/SMARCA4 subunits, resulting in alternative increase of SMARCC1/SMARCA4 binary complex-facilitated gene expression essential for 18S rRNA processing and ribosome biogenesis ( Figure 8J), including BYSL, 24 NOP58 25 and RRP9. 26 Previous investigation reveals that BYSL is enhanced in breast cancer, intestinal adenocarcinomas or gastric cancer, and may promote cell proliferation by facilitating 18S rRNA processing. 27 Meanwhile, knockdown of BYSL decreases the proliferation of hepatocellular carcinoma cells. 27 NOP58, a crucial part of the box C/D snoRNP complex, participates in pre-rRNA cleavage and maturation. 25 In eukaryotes, RRP9 binds to U3 box C/D snoRNA, which is necessary for 18S rRNA processing and 40S subunit assembly, while genetic depletion of RRP9 inhibits early cleavage of primary pre-rRNA transcript and production of 18S rRNA. 26 By gain-and loss-of-function assays, we found that CNBP possessed oncogenic properties via repressing SMARCC2 activity, indicating its potential value in targeting therapeutics of malignancies.
KPNB1 is the first identified nuclear transport factor, 28 which imports a large amount of transcriptional regulators into nucleus. 28 Previous studies have shown that KPNB1 expression is elevated in transformed ovarian cells, cervical cancer, ovarian cancer and breast cancer. 28 During nuclear transport process, karyopherin protein usually requires Ran to provide energy, 28 while blocking RanGTP binding to KPNB1 via a small molecule inhibitor (IPZ) is efficient for treatment of prostate cancer. 12 In this study, we discovered that KPNB1 continuously imported CNBP into nucleus of NB cells, while administration of IPZ restrained this process and suppressed the aggressive behaviours of NB cells.
Phase separation, a compartmentalisation allowing for high local concentrations of proteins and nucleic acids, is crucial for gene transcription, protein homeostasis or signal transduction. 29 For example, phase separation of polycomb repressive complex 1 and autism susceptibility candidate 2 activates gene expression, 30 while β-catenin condensates are essential for Wnt signalling. 31 Meanwhile, the functions of c-Myc or p27 are disrupted by LLPStargeting small molecules. 32,33 Recent evidence shows that phase separation contributes to initiation and aggressiveness of human cancers. 29 Herein, our results indicated that CNBP-SMARCC2 condensates assembling through LLPS were essential for ribosome biogenesis in tumours. In humans, there are 15 SWI/SNF subunits that are assembled into canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF) or non-canonical BAF (ncBAF) complex. 34 In these three complexes, homo-and heterodimers of SMARCC1 and SMARCC2 serve as initial core, 35 while one of two mutually exclusive catalytic subunits, SMARCA4 or SMARCA2, exerts ATPase activity during nucleosome remodelling, gene transcription, DNA repair and replication. 34,36,37 Mutations, translocations or deletions of SWI/SNF subunits are documented in 20% of malignancies, including germline mutations or copy number alterations of SMARCA4. 38 As a key epigenetic regulator, SMARCA4 affects the levels of c-Myc or cyclin D1 in acute leukaemia, 14 lung cancer 15 or colorectal carcinoma. 16 In this study, we found that neither CNBP, SMARCC2, SMARCC1 nor SMARCA4 affected the levels of c-Myc or cyclin D1 in NB cells. In line with our findings, no changes in c-Myc expression were noted in NB cells upon silencing of SMARCA4. 39 Instead, we demonstrated that in a form of phase separation, CNBP interacted with and dissociated SMARCC2 from binding to target gene promoters, triggering the oncogenic roles of SMARCC1/SMARCA4 binary complex in NB progression.
Macrophages are mainly divided into M1 and M2 subtypes, while M2 macrophages exert oncogenic roles. 40 Within microenvironment, tumour cells are able to recruit  and differentiate M2-type macrophages via secreting tumour necrosis factor. 41 As a return, M2 macrophages facilitate tumour proliferation or invasion by activating Toll-like receptor 4/signal transducer and activator of transcription 3. 42 In this study, we found that NB cellderived EVs were able to mediate delivery of 18S rRNA into monocytic THP-1 cells, resulting in differentiation of M2 macrophages. In addition, CNBP repressed the SMARCC2 activity to facilitate rRNA processing and ribosome biogenesis of tumour cells, leading to increase of M2 macrophage polarization. We further developed cellpenetrating peptides blocking the phase separation and interaction of CNBP with SMARCC2, and achieved preferable therapeutic effects on NB progression, suggesting the essential roles of CNBP/SMARCC2 axis in ribosomal biogenesis, M2 macrophage maturation and tumour progression.

CONCLUSIONS
In conclusion, we have shown that CNBP is up-regulated and linked to poor outcome of NB. After nuclear entry mediated by KPNB1, CNBP inhibits the activity of SMARCC2/SMARCC1/SMARCA4 subunits via binding to SMARCC2, leading to alternative increase of SMARCC1/SMARCA4 binary complex-facilitated gene expression essential for ribosome biogenesis and NB progression. In addition, CNBP is able to facilitate EVmediated delivery of 18S rRNA from tumour cells to monocytes, leading to M2 macrophage polarization. Blocking interaction between CNBP and SMARCC2 via cellpenetrating peptide inhibits NB progression. This work broadens current literature regarding the functions of SWI/SNF subunits in controlling ribosome biogenesis, and suggests that KPNB1/CNBP/SMARCC2 axis-mediated ribosome biogenesis and M2 macrophage polarization is a valuable therapeutic target for tumours. Additional investigation is necessary to elucidate the therapeutic effects of targeting CNBP/SMARCC2 in TH-MYCN transgenic or immunocompetent mice, while quantitative proteomics are helpful in exploring additional CNBP protein partners in tumour progression.

Cell lines and culture
The

EV isolation and transfer assay
Conditional media of tumour cells were filtered using 0.22 µm filters, and treated by ultracentrifuging at 100 000 g for 90 min. 43 The extracted EVs were measured by transmission electron microscopic observation, particle size assay, as well as western blotting assay utilising primary antibodies against CD9 (ab236630) and CD63 (ab134045, Abcam Inc., Waltham, MA, USA). Following a 30-min labelling by

Reverse transcription and real-time polymerase chain reaction
The RNAiso Plus kit (Takara Bio Inc, Japan) was utilised to isolate total RNAs. Reverse transcription was undertaken with the PrimeScript RT Master Mix from Takara Bio Inc. The FastStart SYBR Green PCR Mix (Roche, Indianapolis, IN, USA) as well as primer sets (Table S7) were utilised for real-time polymerase chain reaction (PCR), with transcript levels measured by the 2 −ΔΔCt method. 44

Northern blot
TRIzol reagent (Sigma) was applied for preparing total RNAs. For the Northern blot, 20 µg of total RNAs were separated via formaldehyde-agarose gel electrophoresis, shifted to the nitrocellulose membrane (Pall Corp., Port Washington, NY, USA), and incubated with biotin-labelled 5′-ITS-1 or 18S rRNA probe (Table S7) for 16−18 h at 65 • C. Blots were thoroughly washed, recognised by an antihorseradish peroxidase antibody, and detected on X-ray films via chemiluminescence reactions (Roche).

Western blotting
The 1× cell lysis solution (Qiagen, Germantown, MD, USA) was applied in preparing the protein.
Western blotting was carried out as documented, 45

Transcriptome sequencing
By utilising the TRIzol reagent (Life Technologies, Inc.), total RNAs were isolated from tumour cells (1 × 10 6 ). At Wuhan SeqHealth Technology Co., Ltd. (China), transcriptome sequencing and library preparation were undertaken on the Illumina HiSeq X Ten platform. Using the HTSeq v0.6.0 software, 100-bp paired-end raw reads were mapped, and fragments per kilobase of transcript per million incorporation (I) assays indicating the ribosomal subunit assembly, nucleolar NOR dots, nascent protein synthesis and protein production in IMR-32 cells stably transfected with mock or CNBP, and those treated with dimethylsulfoxide (DMSO) or CX-5461 (

Inhibitory peptide synthesis
By using Peptiderive server, 18 inhibitory peptides were designed to prevent interaction between CNBP and SMARCC2. Briefly, the Protein Data Bank (PDB) files of CNBP and SMARCC2 were generated by Phyre2 program, 56 while their docking complex was analysed by ZDOCK program (https://zdock.umassmed.edu). The resulting PDB file was input into Peptiderive server (https://rosie.rosettacommons.org/peptiderive), while size of peptides was specified as 12 aa in length. The domains with highest docking score and corresponding inhibitory peptides were obtained. The cell-penetrating peptides, YGRKKRRQRRR, were derived from transduction domain of Tat protein. 57 Thus, N-terminal biotin-labelled cell-penetrating peptides and C-terminal fluorescein isothiocyanate were chemically conjugated to prepare inhibitory peptides (ChinaPeptides, Shanghai, China). Reversed-phase high-performance liquid chromatography assay was undertaken for evaluating purity of peptides (more than 95%).

5.15
Pull-down using biotin-labelled peptide Using 1× cell lysis solution (Qiagen), cellular proteins were extracted. Then, biotin-labelled peptide was added at 4 • C for an overnight incubation. These peptide-protein complexes were incubated with streptavidin-agarose for 2 h at 4 • C. After thoroughly washing the beads, pulled down proteins were obtained for western blotting.

In vitro phase separation assay
For preparing recombinant proteins, His-tagged CNBP or SMARCC2 construct was transferred into Escherichia coli BL21 strain (Thermo Fisher Scientific, Inc.), while proteins were purified by His-tag Protein Purification Kit (Thermo Fisher Scientific, Inc.). The recombinant proteins were detected by SDS-PAGE and Coomassie blue staining, while their purity was determined by densitometry with ImageJ (https://imagej.nih.gov/ij) program. In brief, after background correction, the density of recombinant protein band was divided by that of whole lane, which was multiplied by 100%. On glass-bottomed dishes, a phase separation test was performed. In droplet formation buffer (1 mmol L −1 dithiothreitol, 10% glycerol, 50 mmol L −1 Tris-HCl, pH 7.5), 40 µmol L −1 of proteins were incubated with solutions containing crowding agent (10% polyethylene glycol-8000) and observed with a Nikon A1R-SI confocal microscope outfitted with oil-immersion lenses.

In vivo phase separation assay
Tumour cells stably expressing mCherry-tagged protein or endogenous protein were grown on glass-bottomed dishes, washed twice with PBS and labelled with Hoechst 33342 (Thermo Fisher Scientific, Inc.) for 10 min. Cells were examined using a Nikon A1R-SI confocal microscope following two PBS washes. The phase separation puncta were defined as visible ones with diameter more than 0.5 µm.

FRAP assay
During in vitro tests, droplets were photobleached with 50% laser power (488 and 561 nm pulses) for 10 s, while time-series photos were taken by using a Nikon A1R-SI confocal microscope outfitted with oil-immersion objectives. In a live-cell imaging chamber, the FRAP test was carried out for in vivo research. Laser pulses at 488 and 561 nm were used to bleach the droplets for 5 s at 50% power, and photobleaching recovery was observed for 1 min. Utilising the FIJI/ImageJ program, fluorescence intensities were normalised to those prior to photobleaching.

Cellular apoptosis and cell cycle assays
For apoptosis detection, tumour cells (5 × 10 5 ) were treated with 5 µL of propidium iodide and Annexin V-FITC (Sigma) in dark for 15 min, and assessed at 488 nm wavelength on a flow cytometer (BD Biosciences, San Jose, CA, USA). In cell cycle assay, tumour cells were treated with 70% ice-cold ethanol and RNase A (2 mg mL -1 ; Sigma). Then, cells were treated by propidium iodide (20 mg mL -1 ; Sigma) for 20 min. A flow cytometer (BD Biosciences) was used to measure cell cycle phases.

Nude mice assays
Nude mice studies were undertaken with Guidelines for the Care and Use of Laboratory Animals set forth by National Institutes of Health, and were approved by Experimental Animal Ethics, Huazhong University of Science and Technology (permission number: 2019-3183). To conduct investigation on subcutaneous xenograft tumours or experimental metastasis, randomised male BALB/c nude mice of 4 weeks at age (n = 5 each group) received injection of tumour cells (1 × 10 6 or 0.4 × 10 6 each mouse). [45][46][47]52,53 In therapeutic investigations, tumour cells (1 × 10 6 or 0.4 × 10 6 ) were administrated into mice's dorsal flanks or tail vein. Two weeks later, inhibitory peptides were administrated via tail vein. The mice were sacrificed when presence of 20% weight loss was recognised as humane endpoint.

Human tissue sample
The investigation using human specimens was authorised by Institutional Review Board of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (permission number: 2017-S242), and were in compliance with Declaration of Helsinki's recommendations. All patients' legal guardians presented their written agreement. None of the cases received any kind of preoperative care, including chemotherapy. From aborted pregnancies, normal dorsal root ganglia were obtained. During surgery, tumour specimens were removed and stored at −80 • C.

Immunohistochemical staining
Immunohistochemistry was undertaken with antibodies against CNBP (sc-515387

Argyrophilic nucleolar organiser region staining
NORs were stained as previously explained. 59 After fixation and rehydration, specimens were incubated with argyrophilic nucleolar organiser region staining solution for 30 min, treated by 5% sodium thiosulphate for 2-5 min, rinsed once again, and observed under a bright field microscopy.

Statistical analysis
For statistical analysis, GraphPad Prism 9 and SPSS 20.0 programs were used, which were obtained from GraphPad Software (San Diego, CA, USA) and SPSS Inc. (Chicago, IL, USA), respectively. The data were presented by mean and standard error of the mean. The cutoffs of gene expression were determined by medium or average levels. The difference was compared by using Pearson chi-square test, one-way analysis of variance or two-sided unpaired Student's t test. Fisher's exact test and Pearson's productmoment correlation assay were used for statistical analysis of overlap or expression correlation. The hazard ratio and difference in survival were evaluated by log-rank test and Cox regression model. A two-sided statistical analysis was used, with p-values less than .05 considered statistically significant.

A C K N O W L E D G E M E N T S
We are grateful for Dr. Arturo Sala for providing vectors. This work was granted by the National Natural Science Foundation of China (82072801, 82173316, 82293663).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no competing interests.

C O N S E N T F O R P U B L I C AT I O N
Written informed consent was obtained from a leagally authorized representative of all patients.